Hitherto, in a treatment for caries (decayed tooth), there has been employed a treatment method involving removing caries by excavation and filling a resin or cement into a tooth after caries excavation, thereby achieving artificial restoration. When a carious cavity caused by caries is large, pulp excavation is halted once in a state in which part of softened dentin is left, and calcium hydroxide is used as a pulp capping material.
Pulp is a vascular connective tissue that fills a cavity called a pulp cavity present inside a tooth, and is a non-mineralized tissue formed from dental papilla derived from mesoderm. On the other hand, dentin is a hard mineralized tissue present around the pulp cavity. Based on the structural features that pulp is a soft tissue and dentin is a hard tissue, both the tissues have conventionally been regarded as quite different tissues. However, in recent years, there has been a growing trend that pulp and dentin are regarded as embryologically and functionally homologous tissues, and in clinical situations as well, as a dentin-pulp complex.
When a tooth is affected by caries, depending on its degree, there is applied a treatment involving protecting pulp without removing it (direct pulp capping), a treatment involving removing only part of pulp (coronal pulp) and preserving radicular pulp (vital pulpotomy), a treatment involving removing the entire pulp and sealing a cavity with a metal or a resin (pulp extirpation), or the like. However, after pulp removal, there arise problems in that dentin weakens because no nutrient is supplied to a tooth and in that no subjective symptom appears when caries progresses again, resulting in exacerbation of caries, because there is no nerve for transmitting pain. Thus, in recent years, it has been considered that it is preferred to preserve pulp as much as possible in order to keep a tooth in a healthy condition. In order to preserve pulp and keep its function, an indirect pulp capping material or a direct pulp capping material is used depending on a damaged condition of dentin and a pathological condition of pulp. The indirect pulp capping material is used in such a condition that dentin is damaged but pulp is not exposed, and the direct pulp capping material is used in a case where pulp is exposed or a case where pulp is partially amputated by a treatment. As the direct pulp capping material, a calcium hydroxide formulation and a formocresol formulation have conventionally been used. However, calcium hydroxide does not have any action of inducing odontoblasts and hence cannot be expected to promote dentinogenesis. Hitherto, there have been reported, as compounds each having an action of promoting dentinogenesis, a bovine blood extract (Patent Literature 1), polysaccharides such as N-acetyl-glucosamine (Patent Literature 2), a bone morphogenetic protein (BMP), and the like.
Calcium hydroxide stimulates odontoblasts in pulp in about three months to promote production of second dentin in a pulp cavity. As a result, healthy dentin is formed between softened dentin and pulp. After that, the softened dentin is removed. However, in this method, calcium hydroxide does not have any action of promoting proliferation of pulp cells and any action of inducing odontoblasts. Hence, a necrosis layer due to a strong alkali is generated in a pulpal surface brought into contact with calcium hydroxide. Further, this method involves causing degeneration and necrosis of a pulpal tissue and regenerating dentin through the subsequent restoration mechanism, and hence involves problems such as along time required for the method, poor efficiency, and indefinite prognosis.
Dentin is a hard tissue that accounts for a large share of a tooth and is present in a state in which it supports the inner pulp and the surrounding enamel and cementum. Dentin is formed by mineralization of organic substrates synthesized and secreted from odontoblasts. Most of the organic substrates are collagens, and the remainder (about 10%) is noncollagenous proteins (NCPs). It is known that dentin sialophosphoprotein (Dspp, hereinafter, simply referred to as “Dspp”), which is the most common among the NCPs, is synthesized by odontoblasts, and then the protein produces dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP).
A variety of research has been advanced on a method of inducing differentiation of pulp cells into odontoblasts and a method of regenerating dentin. As an alternative to calcium hydroxide, there is a disclosure of a pulp capping agent (material) for dentinogenesis including a BMP as an active ingredient (Patent Literature 3). Use of a bone inducing factor such as a BMP was attempted to be applied to production of dentin, focusing on the fact that a component of a mineralization substrate forming dentin is very similar to that of bone. However, the factor such as a BMP has an effect of promoting production of a mineralization substrate by already differentiated odontoblasts, but does not promote differentiation of pulp cells into odontoblasts. Hence, the factor has a very limited effect of inducing odontoblasts in an affected tissue.
A method involving regenerating natural dentin and using the natural dentin for a treatment has been proposed and studied. For example, there is a report that a sample obtained by seeding cultured human pulp cells into a hydroxyapatite-tricalcium phosphate complex powder was transplanted subcutaneously into nude mice, extirpation was carried out after 6 weeks, and formation of a hard tissue was confirmed from a hematoxylin/eosin staining image after the extirpation (Non Patent Literature 1). There is also a report that, when RNA extracted from this sample was evaluated using RT-PCR, mRNA of a differentiation marker for odontoblasts was expressed, in other words, the resultant hard tissue was a dentin-like tissue. In this regard, however, an amount of the formed hard tissue is very small, and a larger amount of dentin needs to be regenerated for clinical applications.
As other technologies relating to dentin regeneration, there is a disclosure of a pulp capping agent (material) for dentin regeneration including a collagen-fixed ethylene-vinyl acetate copolymer saponification product (A) and a binder (B) (Patent Literature 4). Such pulp capping agent (material) for dentin regeneration has an excellent function of securing a scaffold for proliferation of cells each having an ability to regenerate dentin. In addition, as a method of regenerating dentin, there is a disclosure of a method of regenerating dentin involving culturing human pulp cells in the presence of 1,25 (dihydroxy) vitamin D3, dexamethasone, and β-glycerophosphate to achieve differentiation into odontoblasts, and culturing and/or transplanting the cells together with a carrier or the like (Patent Literatures 5 and 6). Further, as another method, there is a disclosure of a pulp capping agent (material) for dentinogenesis using polyphosphoric acid (Patent Literature 7). However, any of the literatures merely discloses a technology for regenerating dentin from odontoblasts, and none of the literatures discloses induction of differentiation of pulp cells into odontoblasts.
It is known that tooth development proceeds through a close interaction between an odontogenic epithelium and an odontogenic mesenchymal tissue. Neural crest-derived mesenchymal cells receive a signal from an ectodermal epithelium tissue and are differentiated into pulp cells. Various growth factors are supposed to be involved in this process. As signals involved in tooth development, for example, there are known a BMP, a fibroblast growth factor (FGF), a Wnt, and a sonic hedgehog (Shh). There is also a report suggesting that transforming growth factor (TGF)-β is involved in differentiation of mature pulp cells into odontoblasts.
A basement membrane is present between an epithelium and a mesenchymal tissue, and one of the constituents for the basement membrane is a proteoglycan. Recent research has clarified that a sugar chain of the proteoglycan of the basement membrane plays an important role in Wnt signaling (Non Patent Literature 2). There is a report that the Wnt induces secretion of Dspp in neural crest cells at the development stage (Non Patent Literature 3). That is, Non Patent Literature 3 reports the Wnt at the tooth development stage, but does not report that the Wnt is involved in differentiation of pulp cells into odontoblasts and regeneration in the already formed tooth.
There is a report that, when a mouse p14 lower molar tooth tissue was confirmed by in situ hybridization using a [α-35S]UTP-labeled RNA probe, Wnt10a was found to be expressed in the basement membrane, and Dspp as an extracellular matrix specific for odontoblasts was found to be expressed in the basement membrane as with Wnt10a (Non Patent Literature 4: see FIG. 1). There is also a report that, when Wnt10a was forcibly expressed in undifferentiated mesenchymal cells C310T1/2 and the cells were cultured with a Matrigel (basement membrane extracts), Dspp mRNA was expressed after 10 days (Non Patent Literature 4: see FIG. 2).
A Wnt signal is conserved across biological species and controls body axis formation and organ formation during early development and cell proliferation and differentiation. It is known that a Wnt signaling pathway includes at least the following three kinds of pathways: 1) a β-catenin pathway, which regulates a transcription factor via intracellular accumulation of β-catenin; 2) a PCP pathway, which controls planar cell polarity via the family of Rho as a low molecular weight G protein; and 3) a Ca2+ pathway, which causes intracellular Ca2+ recruitment via a trimer G protein to activate protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMK II), and the like (Non Patent Literature 5). However, a method of inducing differentiation of pulp cells into odontoblasts to form dentin has not been elucidated yet.